DNA, RNA, and regulatory molecules control gene expression through interactions with RNA polymerase (RNAP). RNA polymerase is an evolutionarily conserved, multisubunit enzyme responsible for utilizing a DNA strand as a template and making a complementary RNA molecule in all known organisms. As such, it is central to all gene transcription and must function for the cell to survive.
The transcription cycle can be divided into major phases: promoter engagement, initiation, RNA chain elongation, and termination. Promoter engagement encompasses several steps: promoter location and recognition by the RNA polymerase holoenzyme (core enzyme complexed with one of several σ factors), initial and reversible binding of RNA polymerase to duplex promoter DNA (closed complex formation), and formation of an open complex in which about 12 bp of DNA, including the transcription start site, are melted. At least four important intrinsic inputs affect promoter engagement: the hexamer centered at position −10 upstream from the transcription start site (−10 region), the hexamer at position −35 (−35 region), the region of DNA between these two elements (spacer region), and a region located between −40 and −60 (the UP element).
The rates at which these steps occur are dictated by both extrinsic and intrinsic interactions. Extrinsic inputs include protein-protein contacts made by activators and repressors that bind in the vicinity of the promoter DNA and can modify the rates of either closed- or open-complex formation or promoter escape. Intrinsic contacts are made by the σ subunit of RNA polymerase to the −10 and −35 regions of the DNA and sometimes by the α-subunit C-terminal domain to the UP element (Ross et al., 1993).
After the open complex has bound the initiating NTPs, it becomes an initial transcription complex and can follow several alternative reaction pathways: (i) the synthesis and release of short (2 to 8 nucleotides [nt]) RNA transcripts (abortive initiation); (ii) reiterative synthesis resulting in homopolymer extensions of the initial RNA transcripts (stuttering); and (iii) release of a, translocation away from the promoter, and formation of a transcription elongation complex (TEC) with loss of upstream DNA contacts, usually when the transcript is 8 to 9 nt in length (promoter escape).
Once RNA polymerase converts from initial transcription complex to TEC (i.e., escapes the promoter), it becomes stably associated with the RNA and DNA chains and can elongate the RNA chain 30 to 100 nt/sec in vivo. Two distinct types of translocations in the TEC must occur to allow this rapid movement; (i) translocation of the RNA 3′ end from position i+1 to i in the active site (the 3′-terminal nucleotide is the index position) as successive nucleotides are added and (ii) translocation of DNA and RNA chains through RNA polymerase (RNA and DNA translocation).
RNA chain elongation is punctuated by certain sites where nucleotide addition is slowed by pause, arrest, and termination signals. Pause signals cause RNA polymerase to isomerize from the rapidly elongating TEC to alternative conformations in which RNA chain extension is reversibly inhibited (by factors of 102 to 104). Termination signals cause the release of RNA and DNA and can be positively or negatively regulated by a variety of extrinsic inputs.
When RNA polymerase encounters a termination signal, RNA polymerase stops adding nucleotides to the RNA, separates the DNA-RNA hybrid, releases the newly synthesized transcript, and dissociates from the DNA template. Three types of termination signals for bacterial RNA polymerase have been described: (i) intrinsic terminators (ρ-independent terminators) which require a stable RNA hairpin formed 7 to 9 nt from the terminated RNA 3′ end and immediately followed by at least 3 U residues, but no extrinsic factors (reviewed in Platt, 1997; Richardson et al., 1996; Roberts, 1996; Uptain et al., 1997); (ii) ρ-dependent terminators, which depend on the presence of ρ factor, a hexameric RNA-binding protein with ATPase activity (reviewed in Platt, 1997; Richardson et al., 1996); and (iii) persistent RNA-DNA hybrid terminators at which pairing of nascent RNA to the template just upstream from the TEC dissociates a complex containing 3′-proximal U-rich RNA (Tomizawa et al., 1997).
E. coli NusA is a 55-kDa acidic protein that interacts with ρ, λ N, and RNA through one or more interaction regions and RNA polymerase through contacts to the α-subunit C-terminal domain and either β′ or β (Liu et al., 1996; Richardson et al., 1996). NusA enhances both pausing and ρ-independent termination in the absence of other cellular or phage proteins, is found in all prokaryotes and archaebaceria sequenced to date, and is essential in E. coli unless ρ activity is reduced by a mutation (Zheng et al., 1994).
A universal feature of RNA polymerases is a regulated conversion from an initiating form that holds the RNA weakly, to an elongating form where the enzyme holds RNA tightly during RNA synthesis, and then back to a terminating form that releases RNA. The molecular basis of the switch from initiation to elongation to termination is unknown. However, conservation from bacteria to humans of RNA polymerase's core subunit composition (β′βα2 in bacteria), amino-acid sequences, three-dimensional structure, and contacts to DNA and RNA suggest that the switch will be similar for all multisubunit RNA polymerases (Zhang et al., 1999; Cramer et al., 2000; Korzheva et al., 2000).
A nascent RNA hairpin can terminate transcription by bacterial RNA polymerase if the hairpin includes the 3-5 nt usually found in the RNA exit channel and disrupts at least one base pair (bp) of the about 8-bp nascent RNA:template DNA hybrid that is present in stable TECs (Nudler et al., 1997; Sidorenkov et al., 1998; Artsimovitch and Landick, 1998; Yarnell and Robert, 1999). Similar RNA hairpins can also pause, rather than terminate, transcription when they form more upstream but near the RNA:DNA hybrid, rather than invade it. Both hairpin-dependent pausing and termination can be enhanced by the universal bacterial protein NusA (Chan and Landick, 1993; Sigmund and Morgan, 1988).
Two models can explain hairpin effects on transcription (Korzheva, 2000; Yarnell and Roberts, 1999; Farnham and Platt, 1980; Yager and von Hippel, 1991; Gusarov and Nudler, 1999; Mooney and Landick, 1999; Davenport et al., 2000; Artsimovitch and Landick, 2000). In the rigid-body model, a pause or terminator hairpin begins forming when only its loop and upper stem have emerged from the exit channel and then pulls RNA through the channel and away from the active site to avoid steric clash with a rigid RNA polymerase as the lower stem pairs. This partially unwinds the RNA:DNA hybrid and moves RNA polymerase forward without nucleotide addition (the hybrid is wedged against the upstream edge of the active-site cleft in a TEC; see FIG. 4A in Korzheva et al., 2000). In the allosteric model, once the hairpin starts to form it instead triggers a conformational change in RNA polymerase that inhibits nucleotide addition in the active site and reduces affinity for product RNA, without necessarily moving the intervening RNA. However, it is unknown which model explains hairpin effects on transcription.
As transcription is central to gene regulation, a better understanding of the transcription process and the cellular factors which interact during transcription could lead to the identification of specific inhibitors of transcription. Thus, what is needed is a method to determine what factors specifically interact with portions of RNA polymerase during transcription. What is also needed is a method to identify agents that specifically inhibit those interactions.